Vector-borne infectious diseases are a major scourge for humanity. Malaria alone, caused by Plasmodium parasites transmitted by the bite of infected female Anopheles mosquitoes, annually infects 250 million people worldwide and kills close to one million, mostly children in sub-Saharan Africa . Past attempts at curbing the disease by the massive use of insecticides and of insecticide-impregnated bed nets have promoted the spread of genetic resistance to a wide range of insecticides across mosquito populations. This is reducing the impact of insecticide-based control methods, and novel approaches to control vector populations are urgently needed to roll back the disease. A similar challenge is posed by the control of Aedes mosquito species transmitting viral diseases including yellow fever, dengue and chikungunya.
Vector control methods that specifically target the desired species represent a valid and environmentally friendly alternative to insecticides. Dating from the 1950s, the sterile insect technique (SIT) is a species-specific control strategy that has been successfully used to reduce the population size of insect pests such as the screw worm and the Mediterranean fruit fly . For mosquito SIT (; reviewed in [4, 5]), large numbers of male insects must be produced in breeding facilities, sterilized by γ-ray irradiation, and released in the field to compete with wild males for mating with wild females. The SIT approach is well suited for Anopheles mosquitoes, as the majority of females mate once in their lifetime and use the sperm stored in their spermatheca to fertilize egg batches produced every time they take a blood meal . This implies that if a female copulates with a sterile male, she will not mate again and will lay only infertile eggs. Repeated releases of sterile males over a given area would, therefore, achieve a local drop in vector populations and a consequent decrease in malaria transmission. However, labour-intensive procedures for selection of male-only populations and a large variability in the efficiency of sterilization have to date posed vast blocks for a massive application of this approach.
Since the turn of the century, vast progress has been made in the generation of molecular and genetic tools for studies on Anopheles mosquitoes. The sequencing of the Anopheles gambiae genome  has allowed the identification of thousands of genes that shape the biology and behaviour of this main malaria vector. These genomic advances, combined with the development of transgenic technologies  to modify the mosquito genome and the possibility of silencing gene expression through RNAi , are facilitating studies on the biological processes that are crucial to the ability of Anopheles mosquitoes to transmit malaria. Importantly, they also offer a basis for novel vector control strategies to complement insecticide treatments. Among the advocated novel approaches are genetic control strategies related to SIT, such as the release of insects carrying a dominant lethal (RIDL), that are currently being developed for the dengue vector, Aedes aegypti[10, 11]. In RIDL, released male mosquitoes carry a transgene that makes their female progeny unviable or infertile. Further alternative strategies propose to alter the genetic make-up of mosquito populations to reduce their vectorial capacity traits, for instance by rendering mosquitoes resistant to human pathogens [12–14]. The introgression of natural genetic traits or of synthetic transgenic constructs interfering with the mosquito vectorial capacity would lead to the replacement of natural disease-transmitting populations with mosquitoes that do not transmit infectious agents, a process that could be enhanced by artificial gene drive systems [15, 16].
Regardless of the final goal of the control programme (population eradication through SIT/RIDL or population replacement), when targeting mosquito vectors male-only populations must be released, since females may (i) contribute to an unwanted increase of the mosquito populations if not sterile, (ii) mate with the released males thereby reducing the efficacy of the trial, and (iii) participate in disease transmission. This underlines the need for high-throughput sexing tools for mosquitoes: males carrying the desired sterility or disease-resistance trait need to be produced on an industrial scale to reach the vast numbers (millions) necessary for a release programme. It is essential that the released males, whether sterile or not, are fully competitive for mating with field females. Therefore, the mechanism utilized for inducing sterility/refractoriness, the mass rearing conditions and the sex sorting procedures must have no impact on overall male fitness.
Proof-of-principle evidence that automated separation of the sexes is achievable in A. gambiae mosquitoes was previously provided using a flow cytometry machine (COPAS®, Union Biometrica) to separate males from females based on the expression of a sperm-specific fluorescence marker in the testis . The usefulness of this system was limited by the fact that male individuals could be identified only during late larval stages, at the onset of sperm development. Here, the full potential of this high-throughput system is achieved by the use of early sex-specific transgenic markers. This system allows the efficient and fully accurate separation of the desired phenotypes at early stages of development. Large larval populations can be sorted into populations of males/females; transgenics/non-transgenics, heterozygous/homozygous, transgenic females/non-transgenic males. Further, the property of the system to quantify transgene copy number offers a new approach for mosquito sexing based on X-linked insertions at any stage of larval development. The sorting procedure has no impact on the mating ability of the resulting adult males. The system, here tested on A. gambiae mosquitoes, could be easily adapted to all mosquito species that are amenable to transgenesis.